The present invention is directed to improved methods and apparati for the production of proteins of interest from a given source material. It should be noted that the production of large quantities of relatively pure, biologically active molecules is important economically for the manufacture of human and animal pharmaceutical formulations, proteins, enzymes, antibodies and other specialty compounds. In the production of many polypeptides, antibodies and proteins, various recombinant DNA techniques have become the method of choice since these methods allow the large scale production of such proteins. The various “platforms” that can be used for such production include bacteria, yeast, insect or mammalian cell cultures as well as transgenic plants or animals. For transgenic animal systems, the preferred animal type is production in dairy mammals, but the transgenics platform technology also contemplates the use of avians or other animals to produce exogenous proteins, antibodies, or fragments or fusions thereof.
Producing recombinant proteins involves transfecting host cells with DNA encoding the protein of interest and growing the host cells, transgenic animals or plants under conditions favoring expression of the recombinant protein or other molecule of interest. The prokaryote—E. coli has been a favored cell culture host system because it can be made to produce recombinant proteins in high yields. However, E. coli are often unable to produce complex or large molecules with proper tertiary folding and resulting in lower or aberrant biological activity.
With improvements in the production of exogenous proteins or other molecules of interest from biological systems there has been increasing pressure on the biotechnology industry to develop new techniques to enhance the volume of production while simultaneously making it more efficient and cost effective in terms of the purification and product recovery. That is, with new products, and larger volumes of known products there is substantial interest in devising methods to bring these therapeutics, in commercial volumes, to market quickly. At the same time the industry is facing new challenges in terms of developing novel processes for the recovery of transgenic proteins and antibodies from various bodily fluids including milk, blood and urine.
Filtration technologies have been major tools in food processing for more than 25 years. The food preparation industry represents a significant part of the filtration and clarification industry world-wide. The main applications of filtration processes are in the dairy industry (whey protein concentration, milk protein standardization, etc.), followed by beverages (wine, beer, fruit juices, etc.) and egg products. Among the very numerous applications of the current invention on an industrial scale, the clarification of fruit, vegetable and sugar juices by microfiltration also allow the flow dynamics to be both simplified and to enhance the final product quality.
With large scale production it is typically the case that there are more complex problems. In addition, there are further challenges imposed in terms of meeting product purity and safety, notably in terms of virus safety and residual contaminants, such as DNA and host cell proteins that might be required to be met by the various governmental agencies that oversee the production of biologically useful pharmaceuticals.
Several methods are currently available to separate molecules of biological interest, such as proteins, from mixtures thereof. One important such technique is affinity chromatography, which separates molecules on the basis of specific and selective binding of the desired molecules to an affinity matrix or gel, while the undesirable molecule remains unbound and can then be moved out of the system. Affinity gels typically consist of a ligand-binding moiety immobilized on a gel support. For example, GB 2,178,742 utilizes an affinity chromatography method to purify hemoglobin and its chemically modified derivatives based on the fact that native hemoglobin binds specifically to a specific family of poly-anionic moieties. For capture these moieties are immobilized on the gel itself. In this process, unmodified hemoglobin is retained by the affinity gel, while modified hemoglobin, which cannot bind to the gel because its poly-anion binding site is covalently occupied by the modifying agent, is removed from the system. Affinity chromatography columns are highly specific and thus yield very pure products; however, affinity chromatography is a relatively expensive process and therefore very difficult to put in place for commercial operations.
In both the biotech industry and in industry ultrafiltration has traditionally been used for size-based separation of protein mixtures wherein the ratio of the protein molecular masses have to be at least around 10 to 1. This has been a limiting factor in many industrial applications throughout industry and in particular in the recovery of biopharmaceuticals in the milk of transgenic mammals. Significant research has taken place in the optimization of ultrafiltration systems by altering the physiochemical conditions (i.e. pH and ionic strength) to achieve higher selectivities (Van Reis et al. (1997)).
More specifically, depth filtration (DF) and tangential flow microfiltration (MF TFF) are two widely adopted filtration techniques that are related, but differ in their manipulation of functional flow mechanics. Generally, in DF processes, the feedstream is preferably introduced perpendicular to the membrane surface. Substances smaller than the membrane pores can become trapped either on the membrane's surface or within the membrane matrix, whereas the filtrate passes through the membrane. Sometimes referred to as “dead-end” or “depth” filtration, DF is commonly used in applications such as clarification, prefiltration, sterile filtration and virus removal. Additionally the majority of depth filters used in the pharmaceutical industry are disposable in nature.
Alternatively, with MF TFF processes, the feedstream is introduced parallel to the membrane surface, resulting in a continuous sweeping of the filtration source material. Under optimal conditions, substances smaller than the membrane's pores escape as filtrate or permeate, and larger particles are retained as retentate. Because of MF TFF's sweeping action and cross-flowing process stream, TFF-based techniques are less prone to fouling than the DF processes of the invention, in which separated particles can accumulate either on or in the membrane. TFF systems exhibit predictable performance characteristics, reliability, and ability to process “difficult” feed streams—all of which have contributed to establishing this platform as the preferred separation method for many biopharmaceutical applications. TFF systems and membranes are not disposable, membranes are cleaned between batches and reused. For these reasons MF TFF systems are frequently used to separate small molecules (1-1000 kD) from larger particulates (1 um-10 um). However, the energy and cleaning associated with the use of MF TFF can often make its use in large volume enterprises impractical.
As mentioned, purifying a recombinant protein from milk is technically complex and expensive. The purification process must be reproducible, involving as few labor-intensive steps as possible, and maximize the yield of the target protein as measured by its biological activity. An ideal purification process optimizes yield, keeping manufacturing costs low.
Clearly then, there remains a need for the development of additional large scale processes for the optimal purification of proteins out of transgenic milk or host cell culture systems which address the relevant quantitative and qualitative issues. The present invention addresses and meets these needs by disclosing a purification process which, in part, relies upon a selective precipitation and depth filtration step which facilitates removal of vast quantities of contaminating/impure compounds, enhancing effectiveness, reducing cost and speeding up processing from a given feedstream.
According to the methods of the current invention improvements have been made to optimize conditions in order to increase the potential size exclusion properties. Various particulates in milk, such as casein and fat, are micelles. These micelles can be manipulated by buffer conditions and be forced to increase or decrease in size. This manipulation of buffer is used to increase the separation efficiency of the depth filtration process. These processes make possible the development of high-performance depth filtration (DF) from various feedstreams including milk. One molecule of interest that can be purified from a cell culture broth or a transgenic milk feedstream is human recombinant antithrombin. Other molecules of interest include without limitation, human albumin, alpha-1-antitrypsin, antibodies, Fc fragments of antibodies and fusion molecules wherein a human albumin protein acts as the carrier molecule. The resulting DF system is employed through the current invention to improve clarification and fractionation efforts even from the levels achieved by TFF.